Supercritical co2 liquorice extract anti-microbial and anti-inflammatory isolates and products made there from

ABSTRACT

Described herein, are compositions comprising, inter alia, a supercritical CO 2  extract of the root of the plant  Glycyrrhiza  sp., and methods of making and using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.12/150,920, filed on May 1, 2008, which is a continuation-in-partapplication of U.S. application Ser. No. 11/799,524, filed on May 2,2007, both of which are incorporated herein by reference.

BACKGROUND

Extracts from the root of plants of the genus Glycyrrhiza (Glycyrrhizasp.), are used for flavorings, the extract imparting the flavor oflicorice (liquorice), among other purposes. Current extraction methodsuse a hot water extraction method. This method preferentially isolateshydrophilic components of the plant root. The major single componentisolated with the prior art extraction methods is glycyrrhizic acid.Glycyrrhizic acid is used predominately as a sweetener and a flavoring.Prior art extraction methods remove little or none of the hydrophobic orlipophilic components of the plant root.

Other prior art methods use organic solvents in the extraction process.These processes remove more of the lipophilic components of the plantroot but have major environmental concerns connected with the use of theorganic solvents. Furthermore, both of the prior art extractionprocesses (hot water and organic solvent) have significant problems interms of, for example, waste generation and elimination. Organic solventextracts have, by their very nature, the problem of spent organics todispose of or recycle. Hot water extraction processes are preferred atextracting hydrophilic compounds are not effective in extractinglipophilic compounds. Additionally, the spent water must be treatedbefore being put back into the water supply of the environment.

Various components of extracts of Glycyrrhiza sp. have been shown tohave anti-inflammatory, antibacterial and anti-adherent effects.However, the effects seen by prior art extracts have been inconsistent.For example, in one study it was reported that incorporation of0.25-0.5% of glycyrrhizic acid (a major component of licorice extractsmade by prior art extraction methods) was ineffective in the removal orprevention of plaque build up (Goultschin, et al., J. Clin. Periodontol.18:210-212, 1991).

Therefore, what is needed is a method for the extraction of the root ofGlycyrrhiza sp. that produces less waste and causes a lesser impact onthe environment than prior art extracts while at the same time producingan extract that is more effective in providing anti-inflammatory,antibacterial and anti-adherent properties by isolating a greaterpercentage of the total available lipophilic components of the plantroot and, in particular, a greater total percentage of the availableprenylated flavonoids of the plant root.

SUMMARY

Some embodiments of the present invention provide methods of treating,reducing, preventing, or ameliorating oral malodor (halitosis). In someembodiments the methods comprise administering an oral care compositioncomprising a supercritical CO₂ extract of the root of the plantGlycyrrhiza sp. to the oral cavity of a subject in need thereof. In someembodiments, the composition is substantially free of glycyrrhizic acid.In some embodiments, the supercritical CO₂ extract of the root of theplant Glycyrrhiza sp. comprises a prenylated flavonoid. In someembodiments, the supercritical CO₂ extract of the root of the plantGlycyrrhiza sp. comprises a plurality of prenylated flavonoids.

The present invention provides, in one aspect, novel and non-obviousmethods of producing extracts from the roots of Glycyrrhiza sp. In oneembodiment, the method of extraction of the present invention comprisesutilizing a supercritical CO₂ extraction process. Details of the processof the present invention are given below in the Description of thePreferred Embodiments. The methods of the present invention solve theproblems of the prior art extraction methods by greatly reducing oreliminating the need for organic solvents during the extraction processand providing an extract that is rich in prenylated flavonoids (e.g.,isoflavans, isoflavonol, flavanone, isoflavone) while also beingvirtually void or void of glycyrrhizic acid (e.g., with no detectableglycyrrhizic acid). The methods of the present invention also extractlipophilic components while extracting very little of the hydrophiliccomponents. The method of the present invention also greatly reduces oreliminates the production of a waste water stream as a result of theextraction process.

The present invention further relates to the extracts made from theextraction processes of the present invention. In one embodiment, theextract of the present invention comprises an extract from G. uralensiscomprising predominately licoricidin and licorisoflavan A(5-O-methyllicoricidin) as the lipophilic components. No detectableglycyrrhizic acid, a hydrophilic component of the plant root, isrecovered with the extraction process of the present invention. Both,licoricidin and licorisoflavan A are prenylated isoflavans, a particulartype of flavonoids. For licoricidin, the molecular weight is m/z=424.For licorisoflavan A: molecular weight m/z=438; UV (MeOH): max=283 nm.In one embodiment, the extract of the present invention is isolatedwithout using ethanol as a modifier during the extraction process. Inthis embodiment, the concentration ranges of licoricidin andlicorisoflavan A are approximately 0.5-6.0% for both. In anotherembodiment, the extract of the present invention is produced usingethanol as a modifier during the extraction process. In this embodiment,the concentration ranges of licoricidin and licorisoflavan A areapproximately 2.0-20.0% for licoricidin and 1.0-8.0% for licorisoflavanA. In yet another embodiment, the concentration of glycyrrhizic acid inthe extracts is less than 0.10%. In yet another embodiment, theconcentration of glycyrrhizic acid in the extracts is less than 0.01% or0.001%. Furthermore, the supercritical CO₂ extracts of the presentinvention also comprises significant amounts of fatty acids (plamiticand linoleic acids, in particular, for example).

In further studies it was discovered that certain components of thesupercritical CO₂ licorice extract of the present invention areexceptionally well suited in providing anti-inflammatory andanti-microbial effects. In this regard, licoricidin and licorisoflavan Aprovide the greatest level of anti-microbial effect. Additionally, inthis regard, the present invention contemplates oral and body carecompositions that comprise amounts of licoricidin and licorisoflavan Aeffective in providing anti-microbial and anti-inflammatory effects.

Other species of Glycyrrhiza provide extracts wherein the predominantlipophilic components are prenylated flavonoids although the exactprenylated flavonoids may differ. For example, an extract of G. glabramade by the extraction process of the present invention would have asthe major prenylated flavonoids, glabridin, methylglabridin and glabrol.

When ethanol is used as a modifier in the extraction process of thepresent invention, the ethanol is at a concentration of betweenapproximately 2.0 and 10%. In a preferred embodiment, the ethanolmodifier is between approximately 4.0 and 6.0%. The use of ethanol isnot necessary to produce the extracts of the present invention. Ethanolhelps to extract highest amount of compounds which have less lipophilicproperties than using CO₂ alone.

The present invention also relates to products that are made from saidextracts. Although the present invention is not limited to anyparticular use for the extract of the present invention, in a preferredembodiment, the extract is used in an oral care product. Examples ofsuitable oral care products in which the extract of the presentinvention can be used include, but are not limited to, dentifrices(e.g., toothpaste, toothpaste gels, toothpowders, denture cleaningagents and compounds, mouthwashes and mouth rinses, toothpicks, dentalfloss, chewing gums, pastilles, lozenges, dissolvable tablets, chewabletablets, etc.). Additionally, the extracts of the present invention maybe used in body care products such as, but not limited to, skin creams,skin lotions, sun screen compositions, soaps, baby care products,shaving products, deodorants, shampoos, etc.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a HPLC chromatogram of extracts of Glycyrrhiza uralensisgenerated with A) hot water extraction, B) ethanol extraction, C) ethylacetate extraction, D) Supercritical CO₂ extraction and E) SupercriticalCO₂ extraction with ethanol as a modifier.

FIG. 2 shows the chemical structures of licoricidin and licorisoflavanA, wherein R=H for licoricidin and R=Me for licorisoflavan A.

DETAILED DESCRIPTION

The present invention relates to extracts from Glycyrrhiza sp. that haveless environmental impact than prior art extraction methods and thatextract a greater percentage of the lipophilic components whileextracting fewer hydrophilic components than the prior art methods. Thepresent invention also relates to uses for the extracts of the presentinvention. In another aspect, the present invention relates to anextraction process of Glycyrrhiza sp. that utilizes a supercritical CO₂extraction process.

Extracts may be made from the root of many different species oflegume-like plants from the genus Glycyrrhiza and more particularly fromthe species Glycyrrhiza uralensis. The plants are native to southernEurope and parts of Asia. G. uralensis is used extensively intraditional Chinese medicine. Still, the present invention is notlimited to any specific plant source. Other known species of Glycyrrhizafrom which extracts my be made thorough the processes of the presentinvention include, but are not limited to, Glycyrrhiza acanthocarpa,Glycyrrhiza aspera, Glycyrrhiza astragalina, Glycyrrhiza bucharica,Glycyrrhiza echinata, Glycyrrhiza eglandulosa, Glycyrrhiza foetida,Glycyrrhiza foetidissima, Glycyrrhiza glabra, Glycyrrhiza gontscharovii,Glycyrrhiza iconica, Glycyrrhiza korshinskyi, Glycyrrhiza lepidota,Glycyrrhiza pallidiflora, Glycyrrhiza squamulosa, Glycyrrhiza triphylla,Glycyrrhiza uralensis and Glycyrrhiza yunnanensis.

Supercritical carbon dioxide refers to carbon dioxide with some uniqueproperties. Carbon dioxide usually behaves as a gas in air or as a solidas dry ice. If the temperature and pressure are both increased, it canadopt properties midway between a gas and a liquid. It behaves like asupercritical fluid above its critical temperature (31.1 degreesCelsius) and pressure (73 atm=73.96 bar), expanding to fill itscontainer like a gas, but with a density like that of a liquid. Theactual temperature necessary to create a supercritical CO₂ depends onthe pressure and vice versa. For example, the higher the pressure, thelower the temperature necessary to reach the supercritical state.Likewise, the higher the temperature, the lower the pressure necessaryto reach the supercritical state.

Although supercritical CO₂ has been used in some instances as anextractant for other substances, it has not, to the best of ourknowledge, been used as an extractant for the extraction of Glycyrrhizasp root especially wherein the resulting extract comprised predominatelyprenylated flavonoids (prenylated isoflavans) and specificallylicoricidin and licorisoflavan A from G. uralensis (and, similarly, theprenylated flavonoids glabridin, methylglabridin and glabrol from G.glabra) and was essentially free or void of glycyrrhizic acid. It wasonly through the empirical experimentation that the methods andcompositions of the present invention were conceived and reduced topractice. There were no market pressures or other market considerationsthat would have led one practiced in the art to identify or develop thecomponents of the present invention, modify the components as necessaryto conceive and practice the present invention and to conceive andproduce the products of present invention. It was only after theconceptual reduction to practice of the present invention that anymarket value was recognized.

Some of the environmental benefits of using supercritical CO₂ are asfollows. First, the CO₂ that is used in the process is essentiallyunchanged and can be reused repeatably. Second, there is virtually nowaste stream, i.e., no spent solvent, volatile organic compounds,contaminated waste water, etc., to deal with. Thus, the extract of thepresent invention is environmentally more sound than the extracts of theprior art methods.

The extracts of the present invention are unlike prior art extracts ofGlycyrrhiza sp. The extracts of the present invention are high in thecompounds known as prenylated flavonoids (e.g., licoricidin andlicorisoflavan A). Both of these compounds have anti-inflammatory,antibacterial, anti-adherent and anti-fungal effects. For example, theextract of the present invention has been shown to have significantantibacterial activity against gram-positive bacteria such asStaphylococcus aureus and Streptococcus mutans, gram-negative anaerobicbacteria like Porphyromonas gingivalis and Prevotella intermedia as wellas antifungal activity against Aspergillus niger. Additionally, theextracts of the present invention are void or virtually void ofglycyrrhizic acid when examined by high pressure liquid chromatography(HPLC) (see, Experimental Section, below). Glycyrrhizic acid has beenused a sweetener and flavor in the prior art and is not needed in thecompositions of the present invention.

Licoricidin and licorisoflavan A are two of the components of thesupercritical CO₂ extract of the present invention. They both showedantimicrobial activity (MIC) on Streptococcus mutans at 3.125 g/ml, theprincipal causal agent of carries, and against Prevotella interrmedia atabout 0.78 g/ml and about 1.56 g/ml for licoricidin and licorisoflavanA, respectively and against Porphyromonas gingivalis at 0.39 g/ml and0.78 g/ml for licoricidin and licorisoflavan A, respectively. Prevotellasp and Porphyromonas gingivalis are involved in periodontitis. Bothcomponents showed also excellent activity as anti-inflammatory agents.Thus, it can be seen that the extract in general and these compounds inparticular can excel in their ability to act as anti-inflammatory,antibacterial and as antifungal agents (See, Experimental section,below).

As mentioned above, the extract produced by the process of the presentinvention can be used in many products including oral care products andbody care products for their anti-inflammatory, antibacterial,anti-adherent and antifungal properties. Below, we describe some of theproducts suitable for use with the extract of the present invention.

The Glycyrrhiza sp root (i.e., licorice extract) of the presentinvention may be formulated in a dental floss. For example, the extractsof the present invention may be impregnated within dental floss so thatit comes into contact with the user's teeth during flossing.Concentrations of the extract of the present invention when used in adental floss composition are about 0.1 to 5.0% by weight of the waxmixture used in coating the floss material. Preferred concentrations areabout 0.5 to 3.0% by weight of the wax mixture used in coating the flossmaterial. Dental floss may be used to ensure that the extracts of thepresent invention come into contact with the surfaces of the teeth thatface each other and are hard to reach by some of the other meansdescribed. When licoricidin and licorisoflavan A are used as theantimicrobial agents in the floss of the present invention they can beused alone or in a mixture at total concentrations of about 0.01 wt % to1.0 wt %, at total concentrations of about 0.05 wt % to 0.5 wt % or attotal concentrations of about 0.1 wt % to 0.2 wt %.

In certain embodiments of the present invention, said dental floss ismade as discussed below. One skilled in the art will realize that thereare other methods and compositions for the manufacture of dental flossthat are compatible with the licorice-based composition of the presentinvention.

The meaning of the words, “dental floss(es),” shall be herein understoodto include both dental flosses and dental tapes as well as any othersimilar article. Moreover, the dental flosses and tapes used in thepresent invention may include any suitable or commercially availabledental floss or tape. These flosses and tapes can be fabricated fromeither natural or synthetic sources examples of which include, but arenot limited to, filaments or yarns of high and normal tenacity polymers,GoreTex™, nylons, polyolefins, polyethylenes, polypropylenes,fluorocarbon compounds, polytetrafluoroethylenes, rayons, dacrons,acrylics, acetate polymers, and other plastics alone or in combination.Natural substances may include, but are not limited to, cotton, wool,silk, linen, hemp, soy and other staple fibers alone or in combination.Blends of synthetic-natural fibers can also be used. However, syntheticfilaments tend to fray less than natural fibers.

The tensile strength of floss must be evenly distributed along theirlength. This is the key with fibers—they will break at their weakestpoint, so even keeled tensile strength is the key. Thus, the dentalfloss of the present invention is related to fibers with high tensilestrength while still being usable for cleaning between teeth. The denierof a floss or floss fiber measures it's weight and also to tensilestrength. Denier is defined as the weight of 9,000 meters of uncoatedfloss in grams. There is a direct correlation between the numericalvalue of the denier and the thickness of the strand of floss: if thisvalue increases, the thickness of the strand also increases. Thus,generally, the higher the denier, the higher the tensile strength, allelse being equal. In one embodiment of the present invention, the denierof round floss is about 750 to 950 and for flat floss it is about 950 to1150.

The length, diameter, structure or design of the floss itself is alsonot limited to any specific size, shape, arrangement or configurationand, thus, can be fabricated to suite any specific intention. It can,for example, be composed of a plurality of individual filaments thathave been formed together to give a larger thread having a sufficientlysmall diameter to permit insertion between the teeth. It can alsocomprise a composite multifilament yarn bonded to an extrudedmonofilament or to another multifilament yarn. A single circular, squareor rectangular shaped monofilament thread is also useful. Other suitablevariations are also well known in the art and as such are also useablein the invention disclosed herein.

Binders, for example, are used in the invention disclosed herein to bindor otherwise attach to a dental floss the ingredients herein specifiedby this disclosure. They also provide the ability to alter thefrictional characteristics of dental floss as well as help bind togetherthe individual filaments comprising the floss itself. Moreover, thevarieties used herein are not restricted to any specific types orcompositions and are thus, given great freedom in their formulations,structures or make-ups. Examples of some suitable binders may thereforeinclude, but are not limited to, natural waxes from plants (e.g.,carnauba wax, soy wax and jojoba wax) as well as other water soluble ornon-water soluble wax or wax-like compounds or water soluble ornon-water soluble polymers, soaps, gums, resins (e.g., myrrh andpropolis) and other substances known in the art.

Flosses may also make use of, for example, one or more solubilizingagents. Their function, as such, will be to aid in dissociation.Suitable solubilizing agents may therefore include, but are not limitedto, filtered water, reverse osmosis water, distilled water and deionizedwater, alone or in combination. However, deionized water has been foundpreferable to the others.

Flosses may also utilize, for example, one or more flavoring agents.These preferably comprise oils or extracts derived from plants andfruits such as citrus oils, fruit essences, mint, peppermint oil,spearmint oil, clove oil, oil of wintergreen, anise, sassafras, sage,eucalyptus, marjoram, cinnamon, lemon, orange, banana, cherry, fennel,apple, pineapple, grape, strawberry and blueberry. Those skilled in theart will recognize that such natural flavoring agents may be usedindependently or combined in any sensorially acceptable blend. All suchflavors and flavor blends are contemplated by the present invention.

To foster greater consumer appeal, flosses may also contain, forexample, one or more natural sweetening agents. These may include, butare not limited to, xylitol, glycerol, sorbitol, maltitol, erythritol,sucrose, lactose, dextrose, maltose, dextrin, fructose, galactose andthe like.

Fluorides have in the past been found to help prevent the incidence ofcarious lesions or caries. Caries are caused when teeth demineralize ata rate faster than they remineralize and most demineralization is causedby acid producing dental plaques. Remineralization, however, is promotedby calcium and phosphate, the chief remineralizing agents found also insaliva. Fluoride based compounds, therefore, provide protection fromcarious lesions or caries by acting as a catalyst to speed theprecipitation of calcium phosphate, in the form of a hydroxy apatite,onto or into teeth. However, this is not fluoride's only role. It isalso able to inhibit the activity of some bacterial enzymes and theiracid producing processes, and at extremely high concentrations it canalso kill certain plaque bacteria. Even more important, it tends tobecome incorporated into the apatite, as a fluoridated hydroxy apatiteor “fluorapatite,” creating a mineral that is appreciably lessdissolvable by acid.

Hence, flosses may contain, for example, one or more fluoride basedcompounds. These compounds may also be slightly soluble in water or maybe fully water soluble. They are, however, foremost characterized bytheir ability to release fluoride ions in water and their freedom fromundesired reactions with the floss's other compounds. Among thesematerials are numerous fluoride-based compounds which can compriseinorganic fluoride salts such as soluble alkali metal, alkaline earthmetal salts, and others. When a fluoride compound is employed, theamount used is dependent to a large extent upon the type of fluorinecompound, its solubility, and the final formulation and structureselected. As such, substantial leeway is given to the quantities oramounts used as long as normal formulation and pharmaceutical safeguardsare observed. Consequently, whenever fluoride based compounds are usedwithin the formulations listed below they should amount to no more than0.30 percent (w/w). However, it has been found that setting the maximumrange at 0.24 percent (w/w) is preferable with 0.22 to 0.24 percent(w/w) being the best overall range to use

The licorice supercritical CO₂ extracts (Glycyrrhiza sp root extract) ofthe present invention may also be used with toothpastes, tooth gels andtooth powders. In one embodiment of the present invention, theconcentration of the extract of the present invention used indentifrices ranges from about 0.002 to 2.0% by weight. In a preferredconcentration the range is between 0.02 to 1.0% by weight. Whenlicoricidin and licorisoflavan A are used as the antimicrobial agents inthe dentifrice of the present invention they can be used alone or in amixture at total concentrations of about 0.01 wt % to 1.0 wt %, at totalconcentrations of about 0.05 wt % to 0.5 wt % or at total concentrationsof about 0.1 wt % to 0.2 wt %.

Compositions in the form of toothpastes, gels, liquid gels, denturecleansing liquids and pastes, and the like, will generally comprise abinder or thickening agent. Binders suitable for use herein includecarrageenan (preferred) and/or natural gums such as gum karaya, xanthangum (preferred), gum arabic (preferred) and gum tragacanth can also beused, as well as other agents known in the art. Colloidal magnesiumaluminium silicate or finely divided hydrated silica can be used as partof the thickening agent to further improve texture. Binders/thickeningagents can be used in an amount from about 0.1% to about 15.0%,preferably from about 1.0 to about 12% by weight of the totalcomposition. The actives are fluoride and potassium nitrate, their levelare described in their respective FDA monographs for anti-cavity andsensitivity. Active for gingivitis are still pending the finalization ofthe monograph. The abrasives are calcium carbonate, baking soda orhydrated silica.

Such compositions will typically comprise one or more sweeteners andflavorings. Examples of suitable sweeteners and flavorings for use indentifrices are discussed above. pH balancing agents (e.g., citric acid,NaOH) as well as, surfactants (e.g., sodium lauryl sulfate),preservatives (e.g., benzoic acid, sorbic acid) stabilizers, etc., arealso often used in dentifrice compositions, examples of which are alsoprovided above. The compositions of the present invention may includebotanicals as well.

In addition to the incorporation of licorice extract of the presentinvention, there may also be included in the dentifrice minor amounts,e.g., 0.01 to 2% by weight of other natural ingredients such asantioxidants, preservatives, pH adjusting agents, desensitizing agents,stabilizing agents, gelling agents, flavors, etc. A preferred gelling orbinding agent is one or more of carrageenan or xanthan gum. Preferredflavorings are natural flavorings such as peppermint, spearmint, apricotand cinnamon, etc. For sensitive teeth, the formulation may contain anagent for relieving pain in people with sensitive teeth. A preferredingredient for this purpose is potassium nitrate. Dispersing agents maybe used in the present invention. Preferred dispersants are sodiumlauryl sulfate or saponin containing plant extracts.

It is also desirable to include one or more humectants (moistureretaining agent) material in the toothpaste to keep the composition fromhardening upon exposure to air. Preferred humectants are glycerin andsorbitol. Certain humectants can also impart a desirable sweetness totoothpaste compositions. Liquid dentifrice and mouthwashes can alsocontain a quantity of humectants. Suitable humectants are well known inthe art. When present, humectants generally represent from about 10% toabout 70%, by weight of the compositions of the invention.

Toothpowders may make use of many of the same ingredients as toothpastesexcept that they must be mixed in a dried state (i.e., dry milled) ormixed as a liquid composition and then dried via, for example, variousknown spray drying techniques. Spray drying is described as when aliquid form of a composition is sprayed as a mist into a hot, drychamber wherein the aqueous portion of the mist is evaporated by the dryheat of the chamber leaving only the dry constituents of the compositionin a powdered form. The powdered form of the composition has a moisturecontent of between about 0.1% to 5%. Toothpowders then rehydrate uponuse either by the addition of water (e.g., with water applied by theuser) at the time of use or by the user's saliva.

Another aspect of the present invention is dissolvable denture cleaningtablet comprising the Glycyrrhiza sp. supercritical CO₂ extract of thepresent invention. Still another aspect of the present invention is adissolvable denture cleaning tablet comprising one or both oflicoricidin and licorisoflavan A isolated, for example, from thesupercritical CO₂ extract of the present invention.

Such tablets may be solid, layered, have a liquid filled center, etc.The tablet should dissolve completely or nearly completely in an aqueoussolution in less than about one hour and, preferably, in less than about15 minutes. The tablet may be effervescent but need not be.

Water-soluble, denture cleanser tablets are made by first preparing anddrying a granulation of ingredients using a suitable binder in an, forexample, anhydrous alcohol solution. The resulting granular mixture isthen dried to a moisture content of 0.4% or less, screened, and thenthoroughly blended with the desired active ingredients along withbinders, fillers, extenders, dyes, flavors, lubricants and the like in asuitable blending apparatus. The final blends are fed into a punch anddie tableting press where it is compacted into tablets. Thereafter thetablets so formed are released and packaged.

Typically, the mouthwashes and rinses of the present invention comprisethe Glycyrrhiza sp. supercritical CO₂ extract of the present inventionin, for example, a water/glycerin solution and may additionally compriseone or more of flavor, humectant, sweetener, emulsifier (e.g.,poloxamer) and colorant. Still another aspect of the present inventionis a mouthwash comprising one or both of licoricidin and licorisoflavanA isolated, for example, from the supercritical CO₂ extract of thepresent invention.

Mouthwashes can include glycerin at a level of from 0 to 60%, preferablyfrom 0 to 30% by weight. The pH value of such mouthwash preparations isgenerally from about 3.5 to about 8.0 and preferably from about 4.0 toabout 7.5. A pH greater than 8.7 would result in an unpleasant mouthfeel. The oral liquid preparations may also contain surface activeagents, i.e., surfactants, in amounts up to about 5.0% and morepreferably between about 0.5 to 2.0%; and fluoride-providing compoundsin amounts up to about 2.0% by weight of the preparation and morepreferably about 0.0442% for sodium fluoride or 0.025% of the fluorideion.

The mouthwashes and mouth rinses of the present invention may alsocomprise one or more alcohols. For example, ethanol.

The chewing gum compositions that comprises the Glycyrrhiza sp.supercritical CO₂ extract of the present invention may be in the form ofa conventional chewing gum or any other product form which is suitablefor chewing. Still another aspect of the present invention is a chewinggum comprising one or both of licoricidin and licorisoflavan A isolatedfrom the supercritical CO₂ extract of the present invention. Suitablephysical forms include sticks, squares and dragees (i.e., sugar coatedgum; e.g., Chiclets™). The chewing gum may also have a liquid-filledcenter wherein the liquid center contains the licorice-basedcompositions (See, e.g., U.S. Pat. No. 6,280,780). The chewing gum mayalso be a digestible or dissolvable gum suitable for chewing. A chewinggum is typically retained in the oral cavity for a time sufficient toallow ingredients released to contact substantially all of the dentalsurfaces and/or oral tissues for purposes of oral activity.

The term “carrier materials” as used herein means any safe and effectiveadditional chewing gum components used in the chewing gum compositionsof the present invention. Such materials include elastomers, resins,plasticisers, fats, solvents, waxes, emulsifiers, softeners, bulkingagents, sweeteners, absorbents, orally active metallic ions, cationicmaterial, fluoride ion sources, additional anticalculus agents,antimicrobial agents, buffers, whitening agents, alkali metalbicarbonate salts, thickening materials, humectants, water, surfactants,titanium dioxide, flavoring agents, xylitol, coloring agents andmixtures thereof.

The chewing gum of the present invention is manufactured by methodsknown in the art. For example, the gum base is heated to 45° C. tosoften. The mixer vessel is maintained at 45° C. during entire mixingprocess and additives as described herein as well as the extract of thepresent invention are added and mixed to homogeneity. Then the gummixture is formed into sticks, squares, etc., wrapped and cooled. Ifdesired, the gum is coated in a sugar or sugar-free candy coating.

The licorice composition may also be used in lozenge and pastillecompositions. The carrier for the lozenges and pastilles is convenientlya sugar, such as glucose, lactose or sucrose, or a substantiallynon-cariogenic material, for example mannitol, xylitol, maltitol,erythritol or sorbitol.

In addition to the active ingredient and the carrier, the lozengespreferably contain one or more binders, such as gelatin (a vegetal gum,e.g., Arabic gum, is preferred) or liquid glucose BPC 1963, which intotal may conveniently be present in an amount of from about 0.5 to 10%weight of the lozenge. A preferred range is from about 1 to 5% byweight.

The lozenges may additionally contain a lubricant, such as stearic acidor a stearate such as magnesium stearate to facilitate manufacture ofthe lozenge. When one or more lubricants are present, the total contentthereof in the lozenge is preferably from 0.1 to 5% by weight.

The lozenges may be prepared by conventional lozenge making procedures,for example by admixing the1,3-bis(2-carboxychromon-5-yloxy)-2-hydroxypropane or salt thereof withthe adjuvant, diluent or carrier and compressing the mixture. In apreferred procedure, the bis-chromone and the adjuvant, diluent orcarrier are desirably first granulated together before being compressedinto the lozenge. The granulation step is preferably a wet granulationstep, and a lubricant is desirably added immediately before thecompression step.

Pastilles (sometimes known as jujubes or soft lozenge) are a hard,jelly-like preparation made from a mixture of glycerin, gelatin (orother natural gelifiant) and water, to which the extract of the presentinvention is incorporated. Flavor may be added too. The glyco-gelatinbase may be melted by gentle heating and any ingredients added. Thewarmed base is then poured into moulds and allowed to set. For example,See, www.rpsgb.org.uk/pdfs/mussheet04.pdf.

Typical body care products include, for example, body lotions, skincreams, soaps, hair care products (for example, shampoos andconditioners), sun screens, anti-inflammatory topical agents, depilatorycompositions, shaving products (for example, shaving cream andaftershave lotions), deodorant, baby care, etc. Body care products areproducts used on the external body (e.g., skin, hair, etc.) as, usually,topical treatments.

One practiced in the art knows how to formulate such body care products.For example, U.S. Pat. No. 6,861,062 to Silva, et al., (and incorporatedherein by reference) discloses exemplary formulations and methods formaking skin creams. Herbal compositions for skin care products are alsoknown in the art (see, e.g., U.S. Pat. No. 6,586,018 to Fasano, which isincorporated herein by reference) discloses exemplary formulations andmethods of manufacture.

Compositions and methods of producing shaving products are also known inthe art. For example, U.S. Pat. No. 7,179,454 to Lucas, which is hereinincorporated by reference, discloses several exemplary formulations andmethods of manufacture. U.S. Pat. No. 6,479,043 to Tietjen, et al.,(incorporated herein by reference) discloses exemplary formulations andmethods of manufacture for depilatory agents.

Likewise, formulations and methods for the production of soaps, sunscreen lotions and anti-inflammatory topical treatments are also knownin the art.

Body care products of the present invention, in one embodiment, comprisean extract of the root of the plant Glycyrrhiza sp., comprising anessentially glycyrrhizic acid free extract wherein the major componentsof the extract are prenylated flavonoids and wherein the root wasextracted by a supercritical CO₂ extraction process.

In another embodiment, the extract is from Glycyrrhiza uralensis and themajor components of the extract are licoricidin and licorisoflavan A.

In another embodiment, the extract is from extract is from Glycyrrhizaglabra and the major component of the extract is glabridin.

For example, a deodorant of the present invention, in one embodiment,comprises an extract of the present invention in combination with anantioxidant compound, preferably with an ingredient from the group oftocopherol and its derivatives, butyl hydroxyanisole (BHA), butylhydroxytoluene (BHT), erythorbic acid, propyl gallate, sodiumerythorbate, tertiary butyl hydroquinone (TBHQ), and rosemary extract,more preferably with ascorbic acid and salts thereof. Further, thedeodorant may comprises certain metal salts of an unsaturated hydroxycarboxylic acid, preferably zinc ricinoleate. In a preferred embodiment,the extract of the present invention is at a concentration of about 0.01to 5.0% by weight and in a more preferred embodiment, from about 0.05 to2.0% by weight. When licoricidin and licorisoflavan A are used as theantimicrobial agents in the deodorant of the present invention they canbe used alone or in a mixture at total concentrations of about 0.01 wt %to 1.0 wt %, at total concentrations of about 0.05 wt % to 0.5 wt % orat total concentrations of about 0.1 wt % to 0.2 wt %.

For body lotions, skin creams, soaps, hair care products, sun screens,anti-inflammatory topical agents, depilatory compositions, shavingproducts, baby care products the concentration of the extracts of thepresent invention are at a concentration of about 0.01-2% and morepreferably 0.1-1%. When licoricidin and licorisoflavan A are used as theantimicrobial agents in the body care products of the present inventionthey can be used alone or in a mixture at total concentrations of about0.01 wt % to 1.0 wt %, at total concentrations of about 0.05 wt % to 0.5wt % or at total concentrations of about 0.1 wt % to 0.2 wt %.

Some embodiments of the present invention provide methods of treating,reducing, preventing, or ameliorating oral malodor (halitosis). In someembodiments the methods comprise administering an oral care compositioncomprising a supercritical CO₂ extract of the root of the plantGlycyrrhiza sp. to the oral cavity of a subject in need thereof. In someembodiments, the composition is substantially free of glycyrrhizic acid.In some embodiments, the supercritical CO₂ extract of the root of theplant Glycyrrhiza sp. comprises a prenylated flavonoid. In someembodiments, the supercritical CO₂ extract of the root of the plantGlycyrrhiza sp. comprises a plurality of prenylated flavonoids.

In some embodiments, the plurality of prenylated flavonoids comprises atleast two prenylated flavonoids selected from the group consisting of:licoricidin; licorisoflavan A; glabridin; methylglabridin; glabrol; and5-O-methyllicoricidin. In some embodiments, the major components of thesupercritical CO₂ extract of the root of the plant Glycyrrhiza sp. arelicoricidin and licorisoflavan A.

In some embodiments, the oral care composition is selected from thegroup consisting of: a toothpaste, a denture cleaner, dental floss, atoothpick, mouthwash, mouth-rinse, a pastille, chewing gum, adissolvable tablet, a chewable tablet and a lozenge.

Some embodiments of the present invention provide methods of reducingvolatile sulfur compounds (VSCs) in the oral cavity comprisingadministering any one of the compositions described herein to the oralcavity of a subject in need thereof. In some embodiments, the volatilesulfur compounds which are reduced in accordance with the methodsdescribed herein are selected from hydrogen sulfide, methyl mercaptan,dimethyl sulfide, and dimethyl disulfide.

In some embodiments, the methods described herein reduce hydrogensulfide and methyl mercaptan.

Embodiments of the present invention are further described in thefollowing examples. The examples are merely illustrative and do not inany way limit the scope of the invention as described and claimed.

EXAMPLES Example 1 Extraction Process

The licorice extract used in the following examples and in themanufacture of dentifrices of the present invention was made with anovel and non-obvious supercritical CO₂ extraction process. Theextraction process of the present invention, in one embodiment, was asfollows. The root of Glycyrrhiza uralensis was cut, ground and powderedto create a raw material. The raw material was put into an extractionchamber (the extractor). The extraction chamber was fed with liquid CO₂.The liquid CO₂, brought from the working/storage tank, was cooled andpumped into the extraction chamber at 300 bar, 50° C. with the 5%addition of 96% ethanol. The CO₂ extract was then metered through anevaporator and flowed into a separation tank.

Under supercritical conditions established in the extractor, CO₂ hassolvent properties for lipophilic ingredients whereas CO₂ in the gasphase is without solvent power under the conditions in the separator (60bar, 30° C.). Thus the extract was precipitated and the gas wasregenerated in the separation tank. The CO₂ was then liquefied bypassing through a condenser and recycled to the working tank.

Example 2 Comparison of the Supercritical CO₂ Licorice Extract of thePresent Invention with the Extracts Made by Prior Art Methods

Broth microdilution minimum inhibitory concentration: For anaerobes, 190μL of sterile, pre-reduced Wilkins Chalgren (WC) anaerobe broth with0.075% cysteine were added to the first well of the test series in a 96well plate, followed by 100 μL in each of four additional test wells.Ten μL of filter-sterilized stock solution of licorice CO₂ extract,prepared at 40 mg/mL in ethanol, was added to the first well of theseries. Contents of the first well were mixed thoroughly and seriallydiluted (1:1) with the remaining four wells in the series; 100 μL werediscarded from the final well in the series. Suspensions ofPorphyromanas gingivalis ATCC 33277 or Prevotella. intermedia ATCC25611, diluted to approximately 1×10⁶ cfu (colony forming units) per mL,were added to the test wells in 100 μL quantities. Appropriate growthpromotion and sterility controls were included. Plates were incubated at37° C. anaerobically, until turbidity in the growth control well wasobserved. The MIC (minimum inhibitory concentration) was recorded as thelowest concentration inhibiting growth of the test organism as viewedwith the unaided eye. Streptococcus mutans ATCC 25175 was tested insimilar fashion, with Tryptic Soy Broth (TSB) as the test medium andincubation of plates at 37° C. with 5% CO₂ . Staphylococcus aureus ATCC6538 was tested using Tryptic Soy Broth (TSB) as the test medium andincubation of plates at 37° C. Aspergillus niger ATCC 16404 was tested,with Tryptic Soy Broth (TSB) as the test medium and incubated at roomtemperature (25° C.).

Results were recorded as the mode value from triplicate experiments vs.each bacterium. P. intermedia and P. gingivalis are gram negativebacteria involved in periodontitis. Streptococcus mutans is involved incaries formation. Both supercritical extracts were able to inhibit thegrowth of those 3 pathogenic bacteria at the lowest concentrations ofextract tested. Supercritical licorice extracts of the presentinvention, with or without using or not ethanol as a modifier, were ableto inhibit P. intermedia at a concentration of 12.5 μg/ml. The extractusing ethanol as a modifier was able to inhibit the growth of P.gingivalis at the same level as chlorhexidine. The supercriticallicorice extract of the present invention (with ethanol) was able toinhibit the growth of S. mutans, a cariogenic bacteria, at aconcentration of 12.5 μg/ml. The supercritical licorice extract of thepresent invention without ethanol was only tested to 31.25 μg/ml. See,Table 1A.

TABLE 1A Antibacterial activity against oral bacteria licorice licoriceCO₂— to CO₂—EtOH extract extract (μg/ml) (μg/ml) ChlorhexidinePrevotella 12.5 12.5 3.125 intermedia Porphyromonas 12.5 6.25 6.25gingivalis Streptococcus <31.25* 12.5 1.56 mutans *The lowestconcentration tested was 31.25 μg/ml

Example 3 Antibacterial Activity of Licoricidin and Licorisoflavan A

Licoricidin and licorisoflavan A are two of the components of thesupercritical CO₂ extract of the present invention. They both showedantimicrobial activity on Streptococcus mutans at 3.125 g/ml, theprincipal causal agent of caries and against Prevotella intermedia atabout 0.78 g/ml and about 1.56 g/ml and Porphyromanas gingivalis at 0.39g/ml and 1.56 g/ml for licoricidin and licorisoflavan A, respectively,as shown in Table 1B. Porphyromonas gingivalis and Prevotella sp areinvolved in periodontitis. These concentrations were the minimuminhibitory concentrations (MIC). Both components showed also excellentactivity as anti-inflammatory agents. MIC is defined by those skilled inthe art as the lowest concentration of an antimicrobial agent that willinhibit the visible growth of a microorganism after overnightincubation. Minimum inhibitory concentrations are used by those skilledin the art to monitor the activity of new antimicrobial agents.Chlorhexidine is used here as a positive control and is frequently foundas an antibacterial agent in, for example, mouthwash.

Broth microdilution minimum inhibitory concentration was performed asfollows. Ten μL of filter-sterilized stock solution of pure compound,licoricidin or licorisoflavan A, prepared at 1 mg/mL in ethanol, wasadded to the first well of the series. Contents of the first well weremixed thoroughly and serially diluted (1:1) with the remaining sevenwells in the series; 100 μL were discarded from the final well in theseries. Suspensions of Porphyromanas gingivalis ATCC 33277 orPrevotella. intermedia ATCC 25611, diluted to approximately 1×10⁶ cfu(colony forming units) per mL, were added to the test wells in 100 μLquantities. Appropriate growth promotion and sterility controls wereincluded. Plates were incubated at 37° C. anaerobically, until turbidityin the growth control well was observed. The MIC (minimum inhibitoryconcentration) was recorded as the lowest concentration inhibitinggrowth of the test organism as viewed with the unaided eye.Streptococcus mutans ATCC 25175 was tested in similar fashion, withTryptic Soy Broth (TSB) as the test medium and incubation of plates at37° C. with 5% CO₂.

Results were recorded as the mode value from triplicate experiments vs.each bacterium. P. intermedia and P. gingivalis are gram negativebacteria involved in periodontitis. Streptococcus mutans is involved incaries formation. Both compounds were able to inhibit the growth ofthose 3 pathogenic bacteria.

TABLE 1B Licoriso- Extract Licoricidin flavan A Chlorhexidine (μg/ml)(μg/ml) (μg/ml) (μg/ml) S. mutans 12.50 3.125 3.125 1.56 P. gingivalis6.25 0.39 0.78 6.25 P. intermedia 6.25 0.78 1.56 3.125

Example 4 Biofilm Formation and Viability

A 24 h culture of P. gingivalis (ATCC 33277) in Todd-Hewitt brothsupplemented with hemin and vitamin K (THB-HK) was diluted in freshbroth medium to obtain an optical density at 655 nm (OD₆₅₅) of 0.07.Samples (100 μL) were added to the wells of a 96-well tissue cultureplate containing 100 μL of serial dilutions (0 to 500 μg/mL) of sterileplant extracts in THB-HK. Control wells with no extract were alsoinoculated. After incubation for 48 h at 37° C. under anaerobicconditions, spent media and free-floating bacteria were removed byaspiration using a 26 G needle and the wells were washed three timeswith distilled water. The P. gingivalis biofilms were stained with 0.4%crystal violet (100 μL) for 15 min. The wells were washed four timeswith distilled water to remove unbound crystal violet dye and dried for2 h at 37° C. After adding 100 μL of 95% (v/v) ethanol to each well, theplate was shaken for 10 min to release the stain from the biofilms andthe absorbance at 550 nm (A₅₅₀) was recorded. Assays were run intriplicate and the means±standard deviations of two independentexperiments were calculated. See, Table 2.

The effect of plant fractions on P. gingivalis cell viability wasinvestigated using the tetrazolium sodium3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) reduction assay. Briefly, XTT wasdissolved in PBS at 1 mg/mL and menadione was prepared in acetone at 1mM. The XTT/menadione reagent contained 12.5 parts XTT/1 part menadione.A 48 h biofilm of P. gingivalis was prepared as above and treated withthe plant fractions (0 to 250 μg/mL) for 2 h (anaerobiosis, 37° C.)prior to adding 25 μL XTT/menadione. After 1 h at 37° C., the absorbanceat 490 nm (A₄₉₀) was read using a microplate reader. The licoriceextract of the present invention was able to inhibit the growth of P.gingivalis and also inhibit the formation of P. gingivalis. P.gingivalis was then unable to attach (anti-adherence). When licoriceextract was applied after the biofilm was formed, there was nodesorption of the biofilm but the viability of the biofilm was reducedwhich means that the biofilm was not removed but that the bacteriaforming the biofilm died. See, Table 2.

TABLE 2 Effect of licorice CO₂ extract biofilm formation, adherenceproperties and viability of P. gingivalis. Inhibit P. Bactericidegingivalis Desorption effect on P. biofilm. of P. gingivalis in (anti-gingivalis Growth of P. a biofilm (cell adherent) biofilm gingivalisviability) (μg/ml) (μg/ml) (μg/ml) (μg/ml) Licorice 4.2 — 4.2 8 CO₂Extract

LPS Preparations:

Aggregatibacter actinomycetemcomitans ATCC 29522 (syns; Actinobacillusactinomycetemcomitans) and P. gingivalis ATCC 33277 were grown in theirappropriate culture media. LPS were isolated from these bacterialstrains by a method routinely used in our laboratory.

Treatments of Macrophages:

U937 cells (ATCC CRL-1593.2), a human monoblastic leukemia cell line,was cultivated at 37° C. in a 5% CO₂ atmosphere in RPMI-1640supplemented with 10% FBS and 100 μg/mL of penicillin-streptomycin(RPMI-FBS). Monocytes (2×10⁵ cells/ml) were incubated in RPMI-10% FBScontaining 10 ng/ml of phorbol myristic acid (PMA) for 48 h to inducedifferentiation into adherent macrophage-like cells. Following the PMAtreatment, the medium was replaced with fresh medium and thedifferentiated cells were incubated for an additional 24 h prior to use.Adherent macrophages were suspended in RPMI-10% FBS and centrifuged at200×g for 8 min. They were washed and suspended in RPMI-10% FBS at adensity of 1×10⁶ cells/ml and seeded in a 6 well-plate (2×10⁶ cells/wellin 2 ml) at 37° C. in a 5% CO₂ atmosphere.

The macrophages were treated with the licorice extract at concentrationsup to 250 μg/ml and incubated at 37° C. in 5% CO₂ for 2 h beforestimulation with LPS at a final concentration of 1 μg/ml. After a 24 hincubation (37° C. in 5% CO₂), the culture medium supernatants werecollected and stored at −20° C. until used. Cells incubated in culturemedium with or without licorice extract, but not stimulated with LPS,were used as controls.

Determination of Cytokine Levels:

Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems,Minneapolis, Minn., U.S.A.) were used to quantify IL-113, IL-6, IL-8,and TNF-α concentrations in the cell-free culture supernatants accordingto the manufacturer's protocols. The absorbance at 450 nm was read in amicroplate reader with the wavelength correction set at 550 nm.

Statistical Analysis:

Two-way analyses of variance were performed to compare the means of thedifferent conditions. Differences were considered significant at the0.05 level (P value).

Anti-inflammatory effect. The extracts of the present invention caused asignificant inhibition in vitro of interleukin 1, interleukin 8 andtumor necrosis production by macrophages produced from a U937 cells, ahuman monoblastic leukemia cell line stimulated with LPS ofAggregatibacter actinomycetemcomitans at a concentration of 10 μg/ml andat 5 μg/ml for interleukin 6. These results were confirmed in twoindependent experiments.

Licorice extract caused a significant inhibition in vitro of interleukin6, interleukin 8 and tumor necrosis factor—(TNF—production bymacrophages stimulated with LPS of P. gingivalis at a concentrationvarying from 5-10 μg/ml. These results were confirmed in two independentexperiments.

HPLC Analysis of the Supercritical CO₂ Extract

Samples of the extracts of the present invention and extracts producedby prior art methods were analyzed by high pressure liquidchromatography (HPLC). The specifications for the procedure used were asfollows:

HPLC was performed on a Zorbax Eclipse XDB C-18 (Agilent Technologies,Santa Clara, Calif.) 250×4.6 mm i.d., 5 m, with Zorbax C-18 GuardColumn: (Cat. #HP 990967-902 and HP 820950-925).

Mobile Phase: MeCN (0.05% TFA)-H₂O (0.05% TFA)

0 min. 45 55 3 min. 45 55 25 min. 100 0 25.1 min. 45 55

Conditioning: at least 20 min.

Flow Rate: 1.0 ml/min

Col. Temp: 30° C.

Detector: 280 nm (bw 16, reference 450 nm, bw 100)

Run time: 25 min.

Post time: 5 min.

The results are presented in FIG. 1. The first chromatogram (A) shows anextract with hot water and it contains only hydrophilic compounds. Thepeak eluting after 4.5 min is glycyrrhizic acid. According to the UVspectrum and the retention time, glycyrrhizic acid is only present inthe hot water extract. The second chromatogram (B) is a 100% ethanolextract and the chromatogram (C) is an ethyl acetate extract, both arevery similar in composition, with the exception that B contains morehydrophilic compounds (eluting between 0-4 min.). The major componentsof the CO₂ extract are licoricidin (1, eluting after 16.5 min) andlicorisoflavan A (2, eluting after 21 min.), together with anunidentified flavonoid peak 3 around 18 min. The supercritical extractusing ethanol (EtOH) as a modifier contains more hydrophilic compoundsthat the supercritical extract without modifier, and contains thehighest amounts of licoricidin and licorisoflavan A. Moreover, from anenvironmental point of view, the CO₂ extract uses only a smallpercentage of ethanol (5-10%) to achieve a good balance of hydrophilicand lipophilic compounds. In both supercritical extracts, thelicoricidin and licorisoflavan A are highly dominant.

Licoricidin and licorisoflavan A were isolated from the supercriticalCO₂ extract of the present invention as follows. A quantity of about 433mg of licorice CO₂/EtOH extract was submitted to medium pressure liquidchromatography (MPLC) with a Büchi C-600 MPLC system (Büchi Analytical,Flawil, Switzerland) using an ODS-based (Phenomenex Luna C-18(2), 100 Å)stationary phase using acetonitrile-water (45:55) as solvent and a flowrate of 5 mL/min. Fraction 3 contained licoricidin (32.8 mg), andlicorisoflavan A (21.5 mg) was obtained from fraction 7. The purity oflicoricidin was about 89% and the purity of licorisoflavan A was about91%. In the context of the present invention, the term “isolated” inregards to the licoricidin and licorisoflavan A of the present inventionincludes purities of about 50% or greater, about 60% or greater, about70 wt. % or greater, about 80 wt. % or greater, about 90 wt. % orgreater, about 95 wt. % or greater or about 99 wt. % or greater.

Example 5

In vitro tests to evaluate the ability of the compositions describedherein are conducted. Hydroxyapatite (HAP) disks are incubated in salivaovernight at 37° C. Slurries comprising the compositions beinginvestigated are prepared by dissolving the respective compositions indistilled water at 1:2 ratio by weight. The saliva-coated HAP disks aretreated with 1 mL of slurry for about 30 minutes. HAP disks are thenrinsed with about 5 mL of distilled water a number of times depending onwhether a short-term (e.g. 3 hour) or long-term (overnight) effect isbeing evaluated. The HAP disks are then evaluated for the presence ofvolatile sulfur compounds (hydrogen sulfide, methyl mercaptan, anddimethyl sulfide) using a gas chromatograph (e.g. Tracor 540).

The results of these in vitro tests are described in Tables 3 and 4(below). The data described in Table 3 demonstrates the reduction inVSCs provided by an exemplary method of the present invention whereinthe compositions evaluated contained 0.05% of an extract (as describedherein) after 2 washes; and the data described in Table 4 demonstratesthe reduction in VSCs provided by an exemplary method of the presentinvention after 4 washes. For example, the ability of the methods of thepresent invention to provide a VSC reduction after four washes indicatesthat the methods of the present invention are able to provide a VSCreduction over an extended period of time (e.g. overnight).

TABLE 3 % Inhibition of Composition VSC production I 48 II 50

TABLE 4 % Inhibition of Composition VSC production I 46 II 45

Example 6

A monadic design, single use clinical study was completed that examinedthe ability of peppermint toothpaste containing an extract, as describedherein, to control oral malodour. After a 7-day pre-treatment washoutperiod using a regular toothbrush (Colgate Wave) and a regular fluoridedentifrice (CDC), eleven (11) subjects reported to the test site withouteating, drinking, or performing oral hygiene. The subjects withqualifying VSC baselines (≧300) ate breakfast, were issued the testproduct, and brushed for one minute under supervision. The subjects wereinstructed to refrain from eating or drinking for the next 4 hours. At 4hours post brushing, subjects returned and provided post use VSCsamples.

Mean baseline and 4 hour post use VSC levels are described in Table 5(below). The data described in Table 5 (below) demonstrates that themethods described herein provide a statistically significant reductionin VSC levels for at least four (4) hours after brushing.

TABLE 5 Composition Baseline 4 hours % Reduction in VSCs I 792.2 223.6*71.8* *Statistically significant reduction from baseline VSC levels (p <0.05)

1. A method of treating oral malodor comprising administering an oralcare composition comprising a supercritical CO₂ extract of the root ofthe plant Glycyrrhiza sp.; and an orally acceptable carrier, to the oralcavity of a subject in need thereof; wherein said extract comprises aplurality of prenylated flavonoids; and wherein said composition issubstantially free of glycyrrhizic acid.
 2. The method of claim 1,wherein the plurality of prenylated flavonoids comprises at least twoprenylated flavonoids selected from the group consisting of:licoricidin; licorisoflavan A; glabridin; methylglabridin; glabrol; and5-O-methyllicoricidin.
 3. The method of claim 1, wherein said oral carecomposition is selected from the group consisting of: a toothpaste, adenture cleaner, dental floss, a toothpick, mouthwash, mouth-rinse, apastille, chewing gum, a dissolvable tablet, a chewable tablet and alozenge.
 4. The method of claim 3, wherein the plurality of prenylatedflavonoids is present in the composition at a concentration of 0.002 to10%, by weight.
 5. The method of claim 4, wherein said composition is atoothpaste; and wherein the plurality of prenylated flavonoids ispresent at a concentration of 0.002 to 2%, by weight.
 6. The method ofclaim 2 wherein the major components of said extract are licoricidin andlicorisoflavan A.
 7. A method of reducing volatile sulfur compounds inthe oral cavity comprising administering an oral care compositioncomprising a supercritical CO₂ extract of the root of the plantGlycyrrhiza sp.; and an orally acceptable carrier, to the oral cavity ofa subject in need thereof; wherein said extract comprises a plurality ofprenylated flavonoids; and wherein said composition is substantiallyfree of glycyrrhizic acid.
 8. The method of claim 7, wherein theplurality of prenylated flavonoids comprises at least two prenylatedflavonoids selected from the group consisting of: licoricidin;licorisoflavan A; glabridin; methylglabridin; glabrol; and5-O-methyllicoricidin.
 9. The method of claim 7, wherein said oral carecomposition is selected from the group consisting of: a toothpaste, adenture cleaner, dental floss, a toothpick, mouthwash, mouth-rinse, apastille, chewing gum, a dissolvable tablet, a chewable tablet and alozenge.
 10. The method of claim 7, wherein the plurality of prenylatedflavonoids is present in the composition at a concentration of 0.002 to10%, by weight.
 11. The method of claim 10, wherein said composition isa toothpaste; and wherein the plurality of prenylated flavonoids ispresent at a concentration of 0.002 to 2%, by weight.
 12. The method ofclaim 8, wherein the major components of said extract are licoricidinand licorisoflavan A.
 13. The method of claim 7, wherein the volatilesulfur compounds are selected from: hydrogen sulfide, methyl mercaptan,dimethyl sulfide, and dimethyl disulfide.
 14. The method of claim 13,wherein the volatile sulfur compounds are selected from hydrogen sulfideand methyl mercaptan.